The polymerase chain reaction (PCR) method is commonly used to amplify specific nucleic acid polymer sequences. The procedure involves several sequential steps, including denaturation of DNA into single strands, annealing of oligonucleotide primers to a template DNA sequence, and extension of the primers with a DNA polymerase (Mullis, K. B. et al., U.S. Pat. Nos. 4,683,202, 4,683,195; Mullis, K. B., EP 201,184; Erlich, H., EP50,424, EP 84,796, EP 258,017, EP 237,362; Erlich, H., U.S. Pat. No. 4,582,788; Saiki, R. et al., U.S. Pat. No. 4,683,202; Mullis, K. B. et al. Cold Spring Harbor Symp. Quant. Biol. 51:263 (1986); Saiki, R. et al. Science 230:1350 (1985); Saiki, R. et al. Science 231:487 (1988); Loh, E. Y. et al. Science 243:217 (1988)).
In theory, the number of primer binding sites doubles with each round of PCR replication because each of the synthetic DNA strands in the reaction, including the original strands and those produced from extension reactions in previous cycles, is available to serve as a template for extending annealed primers in the next round of replication. This aspect of the method, coupled with sufficiently abundant oligonucleotide primer molecules, results in synthetic DNA accumulating in a mathematically exponential manner in successive rounds of replication. The steps in PCR can be repeated many times, to provide large quantities (amplification) of the original target sequence encompassed by the oligonucleotide primers. Even one copy of a DNA sequence can be amplified into hundreds of nanograms (ng) of product (Li, H. et al. Nature 335:414 (1988)). PCR is now a widely utilized tool in molecular biology because of its extreme power and robustness and because of the ready availability of synthetic oligonucleotide primers, thermostable DNA polymerases, and automated temperature cycling machines.
The PCR process is quite susceptible to contamination that is caused by the inadvertent transfer of DNA from one amplification reaction mixture into a subsequent reaction mixture. Even the carryover of one full-length nucleic acid polymer that spans both PCR primer binding sites can be enough to cause a false-positive result in a subsequent reaction. Because the quantities of the amplification products can be large, extreme care must be taken to avoid this problem (Kwok, S. and Higuchi, R. Nature 339:237 (1989)). The presently known procedures for avoiding carryover contamination increase the technical difficulty of carrying out a PCR assay and add significantly to an assay's cost.
One technique for reducing the risk of carry-over contamination has been developed. The technique, known as linked linear amplification (also referred to as “LLA”), uses amplification primers that are modified in such a way that they are, or are rendered, replication defective, non-replicable, or blocked. Primers that have an internal blocking group, such as 1,3-propanediol, can support primer extension and can give rise to amplicons but in subsequent rounds of replication those amplicons give rise to amplicons that terminate in the vicinity of the blocking group and therefore contain only a portion of the primer binding site which is not sufficient for primer binding. See, for example, U.S. Pat. Nos. 6,335,184 and 6,027,923. See, also, Reyes et al. Clinical Chemistry 47:1 31-40 (2001); Wu et al. Genomics 4: 560-569 (1989).
Because the amplicons generated from primer extension products in LLA cannot serve as templates for subsequent primer binding and therefore extension, this DNA does not interfere with subsequent LLA amplification reactions. Although in theory LLA may avoid the cross contamination problem, it requires an extraordinary number of reaction cycles and/or primers to achieve suitable levels of amplified products. For example, U.S. Pat. No. 6,335,184 discloses that 1,000 cycles would be necessary in order to generate a yield of 500,500 products, as opposed to 30 cycles with PCR. The number of reaction cycles can be reduced to some extent by including numerous LLA primers in the amplification reactions. However, Reyes et al. suggest that 14 to 18 primers would be needed to achieve amplification yields comparable with PCR. Clinical Chemistry 47:31-40 (2001). Clearly, this is not a practical solution to the carryover contamination problem because designing and synthesizing such a large number of primers is time consuming and expensive and in most cases it simply isn't possible because there are not a sufficient number of primer-binding sites available and the use of such a large number of primers can poison amplification reactions.
Several other approaches have been developed to avoid the long recognized problem of PCR product contamination. These approaches include chemical decontamination, utilizing closed systems, use of ultra-violet irradiated work stations (Pao et al., Mol. Cell. Probes 7: 217-9 (1993)), cleavable primers (Walder et al., Nucleic Acids Research 21:4, 229-43 (1993))), or enzymatic degradation methods (Longo et al., Gene 93: 125-8 (1990)). None of these methods is totally effective and they involve additional processing steps that complicate the amplification method.
Thus, a need exists for new compositions and methods for routinely amplifying nucleic acids such that the problems associated with carryover-contamination are minimized and which provide suitable levels of amplification without using an excessive number of amplification primers. Ideally such compositions could be used in methods that would provide single-stranded nucleic acid products for direct use in diagnostic methods.
Examples of such compositions and methods have been set forth in U.S. patent application Ser. No. 10/377,168 (“Polynomial Amplification of Nucleic Acids”, filed Feb. 28, 2003) and in International application no. PCT/US2003/006293 (WO03074724; published Sep. 12, 2003), each of which is herein incorporated herein by reference in their entirety. Although the compositions and methods set forth in these applications solve many of the problems set forth above associated with the amplification of nucleic acids, further research has led to improvements, set forth herein, in these compositions and methods for the amplification of nucleotide sequences from a nucleic acid polymer in a high complexity nucleic acid sample, such as genomic DNA.